Recent developments in plant molecular biology suggest that methodology for insertion mutagenesis of plant genes can be developed. This proposal describes experiments aimed at determining whether Agrobacterium tumefaciens-mediated plant cell transformation can produce insertion mutations in a higher plant nuclear genome. Arabidopsis thaliana protoplast isolation, culture, and transformation technologies will be developed as the model system for this study. Available mutants in genetic loci governing nitrate uptake and metabolism by A. thaliani will be studied to define conditions that allow chlorate-resistance selection for insertion mutations at these loci in tissue-cultured cells. Kanamycin-resistant transformants will be generated by cocultivation of haploid A. thaliani protoplasts with A. tumefaciens harboring a genetically-engineered, non-oncogenic Ti plasmid-derived vector. Transformed cells will be co-selected for chlorate resistance. Methodology for generating insertion mutations at selected genetic loci will greatly facilitate the direct isolation of many plant genes that are not accessible with available technologies. These genes may provide useful heterologous probes for characterization of similar genes from mammalian tissues. Better understanding of factors that control eukaryotic gene expression will increase the probability that many congenital and environmentally-related human diseases can be effectively treated and/or eliminated. Another long-term objective of this research is to utilize insertion mutagenesis methodology for the isolation and characterization of genes controlling embryo development from unorganized plant tissue cultures. This model system will provide insight into the hormonal and environmental control of developmental processes in both plants and animals.